Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
  • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

• the invention relates generally to molecular separation techniques, and particularly to techniques for identifying oligonucleotides separated by gel electrophoresis.
• Radioactive labels are highly sensitive, and can be readily incorporated into the molecules of interest.
• Fluorescent labels can be detected immediately after application; they are conveniently handled; and they permit the precise localization and quantification of the labeled molecules.
• four sets of fluorescent dyes are used to detect oligonucleotides whenever mixtures of up to four classes of oligonucleotides are separated electrophoretically on a gel.
• Members from each of the following sets of dyes have been found to be spectrally resolvable with respect to members of every other set under the gel electrophoretic conditions described below.
• Set I consists of fluorescein derivatives defined by the formula: wherein A is a linking functionality at the 5 or 6 carbon position which can be used to link the fluorescein moiety of the dye to a complementary functionality on an oligonucleotide, and B is an acidic anionic group, preferably carboxyl or sulfonic acid, and most preferably carboxyl.
• the linking functionality is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinylamine, or succinimidyl carboxylate whenever the complementary functionality is amine.
• the linking functionality is maleimide, or iodoacetamide whenever the complementary functionality is sulfhydryl.
• Set II consists of derivatives of dichlorodimethoxyfluorescein defined by the formula: wherein A and B are defined as above.
• Set III consists of tetramethylrhodamine derivatized with a linking functionality at the 5 or 6 carbon position, as defined by the formula: wherein A and B are defined as above.
• Set 1V consists of rhodamine X derivatives defined by the formula: wherein A' is a linking functionality as represented by A (as defined above) or an acidic anionic group as represented by B (as defined above), and B' is an acidic anionic group whenever A'is a linking functionality and B' is a linking functionality whenever A' is an acidic anionic group. More preferably, A' is sulfonic acid or a linking functionality as represented by A, and B' is carboxyl or sulfonic acid whenever A' is a linking functionality, and B' is a linking functionality whenever A' is sulfonic acid.
• each class of oligonucleotides prior to separation, members within each class of oligonucleotides are labeled with a dye selected from the same set to form dye-oligonucleotide conjugates, such that the members of different classes are labeled with dyes from different sets. That is, each class corresponds to a different one of the sets I, II, III, or IV defined above (also referred to herein as the first through fourth sets, respectively).
• the members of all classes are combined to form a mixture.
• the mixture is then subjected to gel electrophoresis in order to separate the oligonucleotides according to mass, charge, conformation, and/or other properties which form the bases of one or two dimensional electrophoretic separations.
• Oligomeric series with respect to such properties within and among the classes are determined by the relative positions of similarly separated, e.g. bands, of oligonucleotides on the gel. Finally, the dyes attached to the similarly separated oligonucleotides are caused to fluoresce, and the identity of their class is determined by the fluorescence or absorption spectrum of the attached dye.
• Classes of oligonucleotides can arise in a variety of contexts. For example, they can arise as products of restriction enzyme digests.
• classes identified in accordance with the invention are defined in terms of the terminal nucleotides of nucleic acids so that a correspondence is established between the four possible terminal bases and the four sets of spectrally resolvable dyes. More preferably, the classes arise in the context of chemical or enzymatic sequencing of nucleic acids, and most preferably the classes arise in the context of enzymatic sequencing of DNA.
• Necessary conditions for a class to be identifiable in accordance with the invention are (1) that the oligonucleotides of the class be capable of separation by gel electrophoresis, (2) that they be capable of labeling by the dyes of the invention, and (3) that the classes be mutually exclusive in that an oligonucleotide can only be a member of one class.
• the term “spectrally resolvable” means that the fluorescent emission bands of the dyes within a set are sufficiently distinct, i.e. sufficiently non-overlapping, from those of the dyes of every other set such that the classes of oligonucleotide to which the dyes are attached can be distinguished by standard photodetection systems.
• Oligonucleotide as used herein means a single stranded or double stranded chain of DNA or RNA in the size range of about 10-1000 bases in length (if single stranded), or in the size range of about 10-1000 base pairs in length (if double stranded).
• the advantage of these sets of dyes arise from the nature of their spectral properties in gel environments.
• the gel environments suitable for electrophoretic separations cause a shift of about 10-15 nm toward the red in the absorption and emission bands of the dyes of sets I and II. Shifting of the absorption bands significantly increases the efficiency with which the dyes can be excited with 514 nm light, a major emission line of the argon ion laser, the most cost effective excitation source.
• the emission bands of dyes from set I are shifted away from the 514 nm emission line significantly reducing the amount of scattered light collected with the fluorescent signal from these dyes whenever the dyes are illuminated with 514 nm light.
• the method of the invention finds direct application to chemical and enzymatic DNA sequencing techniques for fluorescently labeling oligonucleotides separated by gel electrophoresis.
• the invention includes methods for detecting up to four predefined classes of oligonucleotides that are electrophoretically separated according to mass, charge, conformation, or other property on the same gel.
• the method is accomplished by labeling oligonucleotides of each class with dyes selected from a separate one of the four sets of dyes defined above. Such labeling ensures that each class has a distinct and spectrally resolvable fluorescent label.
• Set I consists of fluorescein mono-derivatized with a linking functionality at either the 5 or 6 carbon position (as determined by the Color Index numbering system).
• Illustrative examples of set I members include fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate (the -5- and -6- forms being referred to collectively as FITC), fluorescein-5- succinimidylcarboxylate, fluorescein-6- succinimidylcarboxylate, fluorescein-5-iodoacetamide, fluorescein-6-iodoacetamide, fluorescein-5-maleimide, and fluorescein-6-maleimide.
• FITC fluorescein-5-isothiocyanate
• fluorescein-5- succinimidylcarboxylate fluorescein-6- succinimidylcarboxylate
• fluorescein-5-iodoacetamide fluorescein-6-iodoace
• Set II consists of 2',7'-dimethoxy-4',5'-dichlorofluorescein mono-derivatized with a linking functionality at the 5 or 6 carbon position (the carbons being identified in accordance with the Color Index numbering system).
• Set II members can be obtained by standard modifications of 2,7-dimethoxy-4,5-dichloro-9-(2',4'-dicarboxyphenyl)-6-hydroxy-3H-xanthen-3-one and 2,7-dimethoxy-4,5-dichloro-9-(2',5'-dicarboxyphenyl)-6-hydroxy-3H-xanthen-3-one (IUPAC notation) disclosed in U.S. Patent 4,318,846. Accordingly U.S.
• Patent 4,318,846 is incorporated by reference.
• the 4' and 5' carboxys of these compounds can be condensed with N-hydroxysuccinimide using dicyclohexylcarbodiimide to form an amine-selective linking functionality, e.g. as illustrated by examples 6 and 8 of the above-referenced patent (Col.28-29).
• Kasai et al. Anal. Chem., Vol. 47, pgs. 34-37 (1975), discloses the basic technique for such condensations. Accordingly Kasai et al. is incorporated by reference.
• Set III consists of tetramethylrhodamine mono-derivatized with a linking functionality at either the 5 or 6 carbon position.
• Illustrative examples of set III members include tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate (the -5- and -6- forms being referred to collectively as TMRITC), tetramethylrhodamine-5-iodoacetamide, tetramethylrhodamine-6-iodoacetamide, tetramethylrhodamine-5-succinimidylcarboxylate, tetramethylrhodamine-6-succinimidylcarboxylate, tetramethylrhodamine-5-maleimide, and tetramethylrhodamine-6-maleimide.
• These exemplary dyes are available commercially, e.g. Molecular Probes, Inc., or can be
• Set IV consists of rhodamine X derivatives having a disubstituted phenyl attached to the molecule's oxygen heterocycle, one of the substituents being a linking functionality attached to the 4' or 5' carbon (IUPAC numbering) of the phenyl, and the other being a acidic anionic group attached to the 2' carbon.
• set IV members include Texas Red (tradename of Molecular Probes, Inc.), rhodamine X-5-isothiocyanate, rhodamine X-6-isothiocyanate, rhedaming K-5-iodaacetamide, rhodamine X-6-iodoacetamide, rhodamine X-5-succinimidylcarboxylate, rhodamine X-6-succinimidylcarboxylate, rhodamine X-5- maleimide, and rhodamine X-6-maleimide.
• Most of these exemplary dyes are available commercially, e.g.
• Molecular Probes, Inc. can be synthesized using standard techniques.
• Texas Red it can be synthesized according to the procedure disclosed in Titus et al.
• Texas Red a Hydrophilic, Red-Emitting Fluorophore for Use with Fluorescein in Dual Parameter Flow Microfluorometric and Fluorescence Microscopic Studies," J. Immunological. Methods, Vol. 50, pgs. 193-204 (1982).
• 5- and 6- carboxy derivatives of rhodamine X can be synthesized using standard techniques, e.g. as disclosed in U. S. Patent 3,932,415, which is incorporated by reference.
• the 5-or 6- carboxyl groups can then be converted into linking functionalities by standard techniques.
• rhodamine X-succinimidylcarboxylate is formed by techniques disclosed in Muller et al., Experimental Cell Research, Vol. 100, pgs. 213-217 (1976). Accordingly, this reference is incorporated by reference.
• the dyes are attached to oligonucleotides using standard procedures, e.g. for a review see Haugland, "Covalent Fluorescent Probes,” in Excited States of Biopolymers, Steiner, Ed. (Plenum Press, New York, 1983), pgs. 29-58, which pages are incorporated by reference.
• Recently several techniques have been developed for attaching reactive functionalities to oligonucleotides making it possible to form covalent dye-oligonucleotide conjugates by condensing the reactive functionality on the oligonucleotide with a linking functionality of a dye.
• the reactive, or complementary, functionality on the oligonucleotides is an amine.
• the reactive amine is attached by way of the linking agents disclosed in copending U.S. patent application Ser. No. 769,170 filed 26 August 1985, entitled "Amino-derivatized Phosphite and Phosphate Linking Agents, Phosphoramidite Precursors, and Useful Conjugates Thereof.” Accordingly this application is incorporated by reference.
• the reactive amine is attached by reacting 2-methoxy-3-trifluoroacetyl-l,3,2-oxazaphosphacyclopentane with the oligonucleotides.
• nucleic acids separated are oligonucleotides.
• the type of gel is polyacrylamide having a concentration (weight to volume) of between about 2-20 percent. More preferably, the polyacrylamide gel concentration is between about 4-8 percent.
• the gel includes a strand separating, or denaturing, agent. Detailed procedures for constructing such gels are given by Maniatis et al., "Fractionation of Low Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98% Formamide or 7 M Urea," in Methods in Enzymology, Vol. 65, pgs.
• oligonucleotides having sizes in the range of between about 20-300 bases have been separated and detected in accordance with the invention in the following gel: 5 percent polyacrylamide made from 25 parts to 1 part acrylamide to bis-acrylamide, formed in a Tris-borate EDTA buffer at pH 8.3 (measured at 25°C) with 48 percent (weight/volume) urea. The gel was run at 50°C.
• the dye-oligonucleotide conjugates on the gel are illuminated by standard means, e.g. high intensity mercury vapor lamps, lasers, or the like.
• the dye-oligonucleotides on the gel are illuminated by laser light generated by a argon ion laser, particularly the 488 and 514 nm emission lines of an argon ion laser.
• argon ion lasers are available commercially which lase simultaneously at these lines, e.g. Cyonics, Ltd. (Sunnyvale, CA) Model 2001, or the like.
• N-(2-(N',N'-diisopropylaminomethoxyphosphinyloxy)-ethyl)-2,2,2-trifluoroacetamide (7.7 g) was distilled (58-59 °C at 0.8 Torr) to quantitatively yield 2-methoxy-3-trifluoroacetyl-l,3,2-oxazaphosphacyclopentane as a colorless liquid.
• the trifluoroacetyl protection group is removed from the linking agent by treatment with concentrated ammonium hydroxide to give 5'-aminoethylphosphate oligonucleotides. Attachment of the dyes to the exposed amino groups is accomplished by standard procedures, such as the ones described in the following examples.
• a DMF solution of FITC (25 microliters at a concentration of 10 mg/ml, e.g. available from Molecular Probes, Inc., Junction City, OR) is added to a solution of 5'-aminoethylphosphate oligonucleotide (an 18-mer)(0.20 micromolar) in water (200 microliters) and 1 M NaHCO 3 /Na 2 HCO 3 buffer, pH 9.0 (25 microliters).
• the resulting solution is stored in the dark for 6 hours or more.
• the reaction is passed through an equilibrated 10 ml Sephadex G-25 (medium)(a trademarked product of Pharmacia Fine Chemicals) column with water.
• the band of colored material eluting in the excluded volume is collected.
• the crude 5'-fluorescein aminoethylphosphate oligonucleotide is purified by polyacrylamide gel electrophoresis or by HPLC (e.g. Perkin-Elmer Series 4, or comparable device) on a Vydac C18 column (No. 218TP54), or the like, in a linear gradient of 10-20% acetonitrile/0.1 M triethylammonium acetate, pH 7.0.
• a DMF solution of TMRITC (10 microliters at a concentration of 20 mg/ml, e.g. available from Research Organics, Inc., Cleveland, OH, or Molecular Probes, Inc., Junction City, OR) is added to a solution of 5'-aminoethylphosphate oligonucleotide (an 18-mer) (0.004 micromole) in water (88 microliters) and 1 M NaHC0 3/ Na 2 C0 3 buffer, pH 9.0 (2 microliters). The resulting solution is stored in the dark for 6 hours or more. The reaction is passed through an equilibrated 10 ml Sephadex G-25 (medium) column with 0.1 M triethylammonium acetate, pH 7.0. The band of colored material in the excluded volume is purified as for FITC.
• the procedure for attaching Texas Red to the 5'-aminoethylphosphate oligonucleotides can be accomplished by following the same procedure as for . TMRITC.
• DDFCS 5'-aminoethylphosphate oligonucleotide (an 18-mer) (0.006 micromoles in 10 microliters of water ) and 1 M NaHC0 3/ Na 2 C0 3 buffer, pH 9.0 (10 microliters). The resulting solution was stored in the dark for 5 hours and worked up as for FITC.
• DNA sequence analysis is highly useful, both scientifically and commercially.
• the two primary techniques for sequencing DNA fragments are chemical methods, e.g., Maxam and Gilbert, Proc. Nat. Acad. Sci., Vol. 74, p. 560 (1970), and enzymatic replication methods, e.g., Smith, Methods in Enzymology, Vol. 65, Grossman and Moldave, eds., pgs. 560-580 (Academic Press, New York, 1980), and Sanger et al., Proc. Natl. Acad. Sci., Vol. 74, pgs. 5363-5367 (1977).
• the method of the invention can be applied with either technique to substitute fluorescent labels for radioactive labels.
• DNA polymerase is a large multi-function enzyme which catalyzes the synthesis of single-stranded DNA.
• the particular kind of DNA polymerase used in the Sanger method is the so-called Klenow fragment of Escherichia coli DNA polymerase I. This fragment possesses the synthetic function of the enzyme.
• Klenow fragment of Escherichia coli DNA polymerase I. This fragment possesses the synthetic function of the enzyme.
• DNA polymerise requires a template, a primer, and a source of deoxyribonucleotides.
• a template is a single-stranded piece of DNA which determines the sequence of nucleotides in the single-stranded piece of DNA synthesized by the DNA polymerase.
• the DNA polymerase moves along the template, and for each nucleotide base thereof, the DNA polymerase attaches the complementary nucleotide to the growing chain of single-stranded DNA.
• a complementary nucleotide base is one associated with a given base in accordance with the base-pairing rule for the formation of double-stranded DNA.
• the base-pairing rule requires that adenosine of one strand always be paired with thymidine of the other strand, and that cytidine of one strand always be paired with guanosine of the other strand.
• the DNA polymerase encounters an adenosine on the template, it adds a thymidine to the chain being synthesized, and when it encounters a cytidine, it adds a guanosine.
• the newly synthesized chain and the complementary portion of the template are in double-stranded form.
• a primer is a fragment of single-stranded DNA.
• the primer provides a starting location for the DNA polymerase to begin adding nucleotides in the synthesis process.
• the primer must be annealed to the piece of single-stranded DNA containing the template so that a section of double-stranded DNA is provided as the starting point for the DNA polymerase.
• Dideoxyribonucleotides are identical to deoxyribonucleotides except that they lack both the 2' and 3' hydroxyl groups on the ribose moiety, instead of just the 2' hydroxyl as with deoxyribonucleotides.
• Dideoxyribonucleotides are sometimes referred to as analogs of deoxyribonucleotides, in that DNA polymerase accepts the dideoxy derivatives in place of the corresponding deoxyribonucleotide in the DNA synthesis process. When such a substitution takes place, synthesis stops because the DNA polymerase has no 3' hydroxyl group on which to attach the subsequent nucleotide.
• a DNA strand to be sequenced is used as a template for Escherichia coli DNA polymerase I.
• a primer is annealed to a piece of single-stranded DNA containing the template, and then it is extended enzymatically to an average of 20 to 300 or more nucleotides in the presence of radioactively labeled deoxyribonucleoside triphosphates, e.g. 32 p-labeled adenosine triphosphate, and the dideoxyribonucleoside triphosphate analog of one of the four nucleotides. That is, four separate reactions are carried out each including a different dideoxy analog.
• DNA chain growth requires the addition of deoxyribonucleotides to the 3'-hydroxyl, incorporation or a dideoxyribonucleotide terminates chain growth. Incorporation of the dideoxy analog in place of the normal nucleotide occurs randomly, so that each of the four reactions generates a heterogeneous population of labeled strands terminating with the same nucleotide, which can be separated electrophcretically according to chain length. That is, four classes of oligonucleotides are established based on the type of terminal dideoxyribonucleoside which is present. A single stranded DNA phage Ml3 is used to clone copies of the DNA fragment to be sequenced.
• the Ml3 DNA is purified and separated into four aliquots. In each aliquot the synthesis or chain growth reaction takes place in the presence of the respective dideoxyribonucleotides.
• primers are synthesized and then labeled by attaching a linking functionality and reacting it with a dye.
• a linking functionality is attached by reacting the primers with 2-methoxy-3-trifluoroacetyl-l,3,2-oxazaphosphacyclopentane to form 5'-(protected)aminoethylphosphate oligonucleotides.
• the protecting groups are removed and a dye of the invention is attached to the deprotected 5'-amine to form dye-primer conjugates.
• the dye-primer conjugates are then used in accordance with Sanger's method, with the exception that oligonucleotides from the four aliquots are mixed together and loaded onto the same electrophoresis lane.
• the relative size of the oligonucleotides and the nature of their terminal dideoxyribonucleotides are determined as bands of homogeneous oligonucleotides travel down the electrophoresis lane and are detected by a fluorimeter or spectrophotometer after illumination.
• the bands are preferably illuminated with both 514 nm and 488 nm laser light, either sequentially or simultaneously.

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